Elevated mutation rates caused by mutator phenotypes can fuel microbial evolution and neoplastic transformation of mammalian cells. New mutations are assumed to arise at a constant rate in each cell division and segregate equally between the resulting cells. If one or both assumptions are false, an expanding mutator population may contain subclones with widely divergent rates of evolution. Testing these assumptions has not been possible in the past because most mutation rate measurements rely on scoring rare mutations in a small target, which requires large populations of cells. Next Generation Sequencing (NGS) technologies, which permit an entire genome to be used to score mutations, allows us to test the fundamental assumptions about mutation rates by sequencing clones derived from sequential cell divisions. Our strategy is to isolate single-cell lineages fro mutator mother yeast cells, including all daughter cells, the first two grand-daughters born to each daughter, and the first great-granddaughter born to the first grand-daughter. We will grow all isolated cells into clones and then sequence the genomes using NGS. From these data, we will determine the fidelity of individual replication cycles and whether replication errors segregate evenly. In work published this year, we monitored the fixation of mutations in just haploid mutator mother cells and found evidence for distinct mutagenic states. Our first specific aim is to monitor the fixation of all replication errors in both daughter and mother cells. We will use the same haploid strain as well as several diploid mutator strains with higher mutation rates. In another publication this year we found mutations that perturb dNTP levels modulate mutator phenotypes. Thus, in our second specific aim, we will investigate whether mutator volatility is due to dNTP pool regulation. Our third specific aim seeks to identify additional factors that may influence mutator volatility through the isolation of antimutator mutations. This proposal will lea to future studies to investigate whether mammalian mutator cells exhibit distinct mutagenic states and whether factors that influence mutator volatility may be used to modulate mutator phenotypes for therapy.